The faithful duplication of the genetic material is arguably the most fundamental of all biological processes. For DNA viruses as for cells, a complex repertoire of proteins mediates DNA synthesis and coordinates replication with recombination and repair. In this proposal, we present our plan for continued investigation of how vaccinia virus, the prototypic poxvirus, accomplishes genome replication. Poxviruses are among the only DNA viruses whose infectious cycle is restricted to the cytoplasm of infected cells. This autonomy from the host nucleus poses unusual challenges, in that the virus must encode a complete replication apparatus and establish dedicated cytoplasmic sites for replication. Poxviruses represent intriguing model systems in addition to being of significant biomedical importance. The impact of our studies is further strengthened by the importance of the viral replication proteins as targets for the development of rational antiviral therapies. During the next funding period, we will utilize genetic, biochemical and cell biological approaches to further elucidate the replication of vaccinia virus by focusing on the following three aims: AIM I: Analysis of the E9/A20/UDG polymerase holoenzyme in replication and repair. We will continue our studies of the DNA polymerase complex, which is comprised of the E9 DNA Pol and a heterodimeric processivity factor consisting of the A20 and D4 proteins. We will focus on understanding the mechanism whereby UDG/A20 confers processivity on Pol. Because D4 is an active uracil DNA glycosylase (UDG), we will investigate how replication-associated base excision repair is accomplished by viral and cellular proteins. AIM II: Analysis of the mechanism of DNA replication, and investigation of the contributions of the D5 primase/helicase, the A50 ligase, and the G5 nuclease to this process. We will revisit the mechanism of poxvirus replication and assess the possible involvement of both leading and lagging strand synthesis. In this context, we will continue our analysis of the essential D5 ATPase, which is likely to have both primase and helicase activity. We will also investigate the roles of the viral DNA ligase and the G5 protein, which has homology to FEN1 nucleases, in the replication and maturation of the viral genome. AIM III: Analysis of the assembly and structure of viral replication factories. Viral replication takes place in dedicated cytoplasmic regions known as replication factories. These factories are delimited by, and associated with, membranes derived from the endoplasmic reticulum (ER). We will test our hypothesis that the abundant H5 protein serves as a scaffold within the factories. Furthermore, we will use a targeted siRNA library to identify cellular membrane-trafficking proteins that contribute to the reorganization of the ER for the purpose of replication factory assembly.